Ku70 senses cytosolic DNA and assembles a tumor-suppressive signalosome

The innate immune response contributes to the development or attenuation of acute and chronic diseases, including cancer. Microbial DNA and mislocalized DNA from damaged host cells can activate different host responses that shape disease outcomes. Here, we show that mice and humans lacking a single allele of the DNA repair protein Ku70 had increased susceptibility to the development of intestinal cancer. Mechanistically, Ku70 translocates from the nucleus into the cytoplasm where it binds to cytosolic DNA and interacts with the GTPase Ras and the kinase Raf, forming a tripartite protein complex and docking at Rab5+Rab7+ early-late endosomes. This Ku70-Ras-Raf signalosome activates the MEK-ERK pathways, leading to impaired activation of cell cycle proteins Cdc25A and CDK1, reducing cell proliferation and tumorigenesis. We also identified the domains of Ku70, Ras, and Raf involved in activating the Ku70 signaling pathway. Therapeutics targeting components of the Ku70 signalosome could improve the treatment outcomes in cancer.


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Figs. S1 to S22 Tables S1 to S3 Fig. S1.Changes in the gene encoding Ku70 are associated with colorectal cancer in humans.(A) Number of cancer cases with respect to alterations in gene encoding Ku70 (XRCC6), determined using The American Association for Cancer Research (AACR) project Genomics Evidence Neoplasia Information Exchange (GENIE) from My Cancer Genome portal.(B) Type of alteration in XRCC6 determined using AACR project GENIE from My Cancer Genome portal.snRNA-seq data (A) and average expression of the gene encoding Ku70 in cells of stromal, immune, and epithelial compartments obtained from normal, polyp, and colorectal cancer samples (B).(C and D).UMAP representation of snRNA-seq data (C) and average expression of the gene encoding Ku70 in cells of stromal compartment obtained from normal, polyp, and colorectal cancer samples (D).(E and F).UMAP representation of snRNA-seq data (E) and average expression of the gene encoding Ku70 in cells of the epithelial compartment obtained from normal, polyp, and colorectal cancer samples (F).The size of the dots denotes the fraction of each cell type expressing the gene encoding Ku70, whereas, color bars denote the average expression of the gene encoding Ku70 in indicated cells.Data are available from the Gene Expression Omnibus (GSE201348).   .The gut microbiota composition is similar between littermate-controlled WT and Ku70 +/-, and Apc Min/+ and Apc Min/+ Ku70 +/-mice.(A to D) Species richness (A), species evenness (B), and Shannon's diversity (C) of bacterial composition and principal-coordinate (PCO) analysis of a Bray-Curtis resemblance matrix, generated from square-root-transformed relative abundances of bacterial operational taxonomic units (OTUs) (D) in the fecal pellets from the colon of untreated (day 0) and AOM-DSS treated (day 80) littermate WT and Ku70 +/-mice.(E to H) Species richness (E), species evenness (F), and Shannon's diversity (G) of bacterial composition and PCO analysis of a Bray-Curtis resemblance matrix, generated from square-root-transformed relative abundances  Heatmap showing differentially phosphorylated proteins after global phospho-proteomic screen on the colon tissue lysate of littermate WT (n=5), Ku70 +/-(n=5), and Ku70 -/-(n=5) mice on the 129 genetic background 14 days after AOM injection.Heatmap was created using the Heatmapper web server, the Average linkage method was used for clustering (see Table S1 for the number of clusters), and the Pearson method was used for the distance measurement.WT 1a, IL-b, IL-2, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-15, keratinocytesderived chemokine (KC), leukemia inhibitory factor (LIF), C-X-C motif chemokine 5 (LIX) and macrophage colony-stimulating factor (M-CSF) in the colon tissue of untreated (day 0) and AOM-DSS treated (day 14) littermate WT, Ku70 +/-and Ku70 -/-mice, determined using multiplex ELISA.Each symbol represents an individual mouse.NS, not statistically significant by one-way ANOVA with Tukey's multiple comparisons test.Data are pooled from three independent experiments and are presented as mean ± s.e.m.Cooccurrence *A positive value for the Log2 Odds Ratio suggests that alterations in these genes co-occur in the same samples, while a negative value suggests that alterations in these genes are mutually exclusive and occur in different samples.

Gene
Fig. S2.The expression of the gene encoding Ku70 changes in a cell type-specific manner during the development of colorectal cancer in humans.(A and B) UMAP representation of

Fig. S3 .
Fig. S3.The expression of the gene encoding Ku70 changes in a cell type-specific manner during the development of Crohn's disease.(A to C) UMAP representation of scRNA-seq data (A) and average expression of the gene encoding Ku70 in stromal, immune, and epithelial compartments (B) and different cell types of stromal, immune, and epithelial compartments (C) obtained from healthy, non-inflamed, and inflamed samples from patients with Crohn's disease.The size of the dots denotes the fraction of each cell type expressing the gene encoding Ku70, whereas, color bars denote the average expression of the gene encoding Ku70 in indicated cells.Data are available from the Broad Single Cell Portal (SCP1884).STR: Stromal cells; EPI: Epithelial cells; IMM: Immune cells; Heal: Healthy samples; NonI: Non-inflamed samples; Infl: Inflamed samples.
Fig. S5.Heterozygous deletion of XRCC6 is commonly found in tumors of patients with colon and rectal adenocarcinoma.(A and B)Percentage of copy number variation (CNV; amplification and/or deletion) in XRCC6 in colon adenocarcinoma (A) and rectal adenocarcinoma (B), determined using Gene Set Cancer Analysis web server.
Fig. S7.The gut microbiota composition is similar between littermate-controlled WT andKu70 +/-, and Apc Min/+ and Apc Min/+ Ku70 +/-mice.(A to D) Species richness (A), species evenness (B), and Shannon's diversity (C) of bacterial composition and principal-coordinate (PCO) analysis of a Bray-Curtis resemblance matrix, generated from square-root-transformed relative abundances of bacterial operational taxonomic units (OTUs) (D) in the fecal pellets from the colon of untreated (day 0) and AOM-DSS treated (day 80) littermate WT and Ku70 +/-mice.(E to H) Species richness (E), species evenness (F), and Shannon's diversity (G) of bacterial composition and PCO analysis of a Bray-Curtis resemblance matrix, generated from square-root-transformed relative abundances Fig. S8.Ku70 does not affect DNA damage during the development of colitis.(A and B)Immunohistochemical staining of 53BP1, β-actin, and DAPI in the colon tissue of untreated littermate WT, Ku70 +/-and Ku70 -/-mice on the 129 genetic background on day 0 (top left), 14

Fig. S9 .
Fig. S9.Ku70 affects the phosphorylation status of proteins in the colon tissue of mice with colitis.Heatmap showing differentially phosphorylated proteins after global phospho-proteomic screen on the colon tissue lysate of littermate WT (n=5), Ku70 +/-(n=5), and Ku70 -/-(n=5) mice on the 129 genetic background 14 days after AOM injection.Heatmap was created using the Heatmapper web server, the Average linkage method was used for clustering (see TableS1for the number of clusters), and the Pearson method was used for the distance measurement.

Fig. S10 .
Fig. S10.Ku70 preferentially activates the ERK signaling pathway during the development of colitis.(A) Densitometric quantification of immunoblots of indicated proteins from the colon tissue lysate of mice at day 14 as shown in Fig. 4C.(B) Immunoblot of the indicated proteins (left) and densitometric quantification (right) on the colon tissue lysate mice on the 129 genetic background at day 0. Each symbol (A and B) and lane (B) represents an individual mouse.Pindicates phosphorylated protein in (B).Data are representative of three independent experiments in (A and B) and are presented as mean ± s.e.m. in (A and B).

Fig
Fig. S11.Ku70 activates the ERK-signaling pathway during the development of intestinal cancer.(A) Immunohistochemical staining of P-ERK, β-actin, and DAPI (left) and quantification of P-ERK-positive area over total tissue area (right) in the colon tissue of mice 80 days after AOM injection.Scale bar, 50μm.(B) Immunoblot of the indicated proteins on the colon tissue lysate of Fig. S12.Ku70 phosphorylates MEK during the development of colitis and colitis-associated colorectal cancer.(A) Immunoblot of the indicated proteins on the colon tissue lysate of littermate WT, Ku70 +/-and Ku70 -/-mice at day 14 (left) and on the colon tissue lysate of littermate WT and Ku70 +/-mice at day 80 (right).(B) Densitometric quantification of the blots as shown in (A).(C and D) Immunoblot of the indicated proteins (C) and densitometric quantification (D) on the colon tissue lysate of mice.(E) Immunoblot of the indicated proteins on the colon tissue lysate of littermate WT, Ku70 +/-and Ku70 -/-mice at day 14 (left) and on the colon tissue lysate of littermate WT and Ku70 +/-mice at day 80 (right).(F) Densitometric quantification of the blots as shown in (E).(G) Immunohistochemical staining of P-ATM, b-actin, and DAPI in the colon tissue of untreated littermate WT, Ku70 +/-and Ku70 -/-mice on the 129 genetic background on day 14 (left) or day 80 (right) after AOM-DSS treatment.Scale bar, 50μm.(H) Quantification of the P-ATMpositive area over total colon tissue area as in (G).Each lane (A, C and E) or symbol (B, D and F) represents an individual mouse or one of the three (proximal, middle, or distal) parts of the colon in (H).P-indicates phosphorylated protein and T-indicates total protein in (A to F). NS, not statistically significant; *P<0.05;**P<0.01***P<0.001;by one-way ANOVA with Tukey's multiple comparisons test (B and F, left panels and H top panel) or unpaired t-test (B, right panel, D and F, right panel and H bottom panel).Data are representative of three independent experiments and are presented as mean ± s.e.m. in (B, D, F and H).

Fig. S14 .
Fig. S14.Ku70 restricts overt cell proliferation during the development of colitis and intestinal cancer.(A) Representative images of immunohistochemical staining of Ki67, b-actin, and DAPI (left) and PCNA, b-actin, and DAPI (right) in the colon tissue of littermate WT, Ku70 +/- and Ku70 -/-mice.Scale bar, 50μm.(B) Quantification of Ki67 (top) and PCNA (bottom) positive area over total tissue area.(C) Representative images of immunohistochemical staining of Ki67, b-actin, and DAPI (top left) and PCNA, b-actin, and DAPI (bottom left) in the colon, and Ki67, b-actin, and DAPI (top right) and PCNA, b-actin, and DAPI (bottom right) in the distal small intestine of 20 weeks old littermate Apc Min/+ and Apc Min/+ Ku70 +/-mice.Scale bar, 50μm.(D) Quantification of Ki67 (top left) and PCNA (bottom left) in the colon, and Ki67 (top right) and PCNA (bottom right) in the distal small intestine of 20 weeks old littermate Apc Min/+ and Apc Min/+ Ku70 +/-mice.Each symbol represents one of the three (proximal, middle, or distal) parts Fig. S15.Ku70 may contribute to controlling intestinal stem cell proliferation via ERK signaling.(A)Immunoblot of the indicated proteins on the cell lysates of mouse intestinal crypts isolated from the colon of 10-week-old littermate Apc Min/+ and Apc Min/+ Ku70 +/-mice.(B) Images of mouse intestinal organoids cultured from stem cells of the colon of 10-week-old littermate Apc Min/+ and Apc Min/+ Ku70 +/-mice at passage 0 (left), passage 1 (middle), and passage 2 (right).Scale bars, 50μm (C) Size (perimeter; au, arbitrary unit) of mouse intestinal organoids as in (B) at passage 0 (left), passage 1 (middle), and passage 2 (right).(D to G) Immunofluorescence staining of Ki67, CD324 (E-Cadherin) and DAPI in mouse intestinal organoids as in (B), at passage 0 (D), passage 1 (E), and passage 2 (F); and quantification (G) of Ki67-positive organoid area over the total organoid area at passage 0 (left), passage 1 (middle), and passage 2 (right).Scale bars, 50μm.(H) Immunoblot of the indicated proteins on the lysates of mouse intestinal organoids left untreated (time 0) or treated with insulin growth factor (IGF)-1 (50ng/ml) for the indicated time.Each symbol represents an individual organoid in (C and G).*P<0.05;**P<0.01;***P<0.001;****P<0.0001by two-way ANOVA with Šídák's multiple comparisons test (C and G).Data are representative from two independent experiments in (A to H) and are presented as mean ± s.e.m. in (C and G).

B
Data are representative of three (A) and two independent experiments in (B) and presented as mean ± s.e.m (B).

Fig. S22 .
Fig. S22.Ku70 does not control MEK-ERK signaling in colorectal cancer cell lines harboring KRAS and BRAF mutations.(A) Electropherogram of DNA from Sanger sequencing of the gene encoding KRAS in HCT116 cells, showing a G>A mutation in codon 13 (c.13G;corresponding to p.G13D).The presence of both a G (black) and A (green) peak in codon 13 indicates a heterozygous mutation (Left).Electropherogram of DNA from Sanger sequencing of the gene encoding BRAF in HT29 cells, showing a C>G mutation in codon 119 (c.119T; corresponding to p.T119S).The presence of both G (black) and C (blue) peaks in codon 119 indicates a heterozygous mutation (Middle).Electropherogram of DNA from Sanger sequencing of the gene encoding BRAF in HT29 cells, showing a T>A mutation in codon 600 (c.600V; corresponding to p.V600E).The presence of both T (red) and A (green) peaks in codon 600 indicates a heterozygous mutation (Right).(B) Immunoblot of the indicated proteins on the cell lysates of serum-starved HEK293T, HCT116, and HT29 cells transfected with an empty vector (EV) or Ku70 plasmid.The letter underneath the black line below the three nucleotides indicates the corresponding amino acid of the codon (A).Data are representative of three experiments in (B).

Table S1 . List of differentially phosphorylated proteins in the colon tissue of mice with colitis Number of clusters (cluster # from)
Higher, higher phosphorylation intensity of a protein in one group as compared to another and/or two other group/s; **Lower, lower phosphorylation intensity of a protein in one group as compared to another and/or two other group/s.